Genomic targets of HOP2 are enriched for features found at recombination hotspots.

HOP2 is a conserved protein that plays a positive role in homologous chromosome pairing and a separable role in preventing illegitimate connections between nonhomologous chromosome regions during meiosis. We employed ChIP-seq to discover that Arabidopsis HOP2 binds along the length of all chromosomes, except for centromeric and nucleolar organizer regions, and no binding sites were detected in the organelle genomes. A large number of reads were assigned to the HOP2 locus itself, yet TAIL-PCR and SNP analysis of the aligned sequences indicate that many of these reads originate from the transforming T-DNA, supporting the role of HOP2 in preventing nonhomologous exchanges. The 292 ChIP-seq peaks are largely found in promoter regions and downstream from genes, paralleling the distribution of recombination hotspots, and motif analysis revealed that there are several conserved sequences that are also enriched at crossover sites. We conducted coimmunoprecipitation of HOP2 followed by LC-MS/MS and found enrichment for several proteins, including some histone variants and modifications that are also known to be associated with recombination hotspots. We propose that HOP2 may be directed to chromatin motifs near double strand breaks, where homology checks are proposed to occur.

The Arabidopsis HOP2 gene has a role in preventing illegitimate connections between nonhomologous chromosome regions.

Meiotic homologous chromosomes synapse and undergo crossing over (CO). In many eukaryotes, both synapsis and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via homologous recombination. In these organisms, two key proteins are recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 assist RAD51 and DMC1 and also are required for synapsis and CO. We have investigated the hop2-1 phenotype in Arabidopsis during the segregation stages of both meiosis and mitosis. Despite a general lack of synapsis during prophase I, we observed extensive, stable interconnections between nonhomologous chromosomes in diploid hop2-1 nuclei in first and second meiotic divisions. Using γH2Ax as a marker of unrepaired DSBs, we detected γH2AX foci from leptotene through early pachytene but saw no foci from mid-pachytene onward. We conclude that the bridges seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Examining haploids, we found that wild type haploids produce only univalents, but hop2-1 haploids like hop2-1 diploids have illegitimate connections stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing repairs that use nonhomologous chromosomes during meiosis. We also found evidence that HOP2 plays a role in preventing illegitimate repair of radiation-induced DSBs in rapidly dividing petal cells. We conclude that HOP2 in Arabidopsis plays both a positive role in promoting synapsis and a separable role in preventing DSB repair using nonhomologous chromosomes. SIGNIFICANCE STATEMENT : The fidelity of homologous recombination (HR) during meiosis is essential to the production of viable gametes and for maintaining genome integrity in vegetative cells. HOP2 is an important protein for accurate meiotic HR in plants. We have found evidence of high levels of illegitimate repairs between nonhomologous chromosomes during meiosis and in irradiated petal cells in hop2-1 mutants, suggesting a role for HOP2 beyond its established role in synapsis and crossing over.

flasher, a novel mutation in a glucosinolate modifying enzyme, conditions changes in plant architecture and hormone homeostasis.

Meristem function is underpinned by numerous genes that affect hormone levels, ultimately controlling phyllotaxy, the transition to flowering and general growth properties. Class I KNOX genes are major contributors to this process, promoting cytokinin biosynthesis but repressing gibberellin production to condition a replication competent state. We identified a suppressor mutant of the KNOX1 mutant brevipedicellus (bp) that we termed flasher (fsh), which promotes stem and pedicel elongation, suppresses early senescence, and negatively affects reproductive development. Map-based cloning and complementation tests revealed that fsh is due to an E40K change in the flavin monooxygenase GS-OX5, a gene encoding a glucosinolate (GSL) modifying enzyme. In vitro enzymatic assays revealed that fsh poorly converts substrate to product, yet the levels of several GSLs are higher in the suppressor line, implicating FSH in feedback control of GSL flux. FSH is expressed predominantly in the vasculature in patterns that do not significantly overlap those of BP, implying a non-cell autonomous mode of meristem control via one or more GSL metabolites. Hormone analyses revealed that cytokinin levels are low in bp, but fsh restores cytokinin levels to near normal by activating cytokinin biosynthesis genes. In addition, jasmonate levels in the fsh suppressor are significantly lower than in bp, which is likely due to elevated expression of JA inactivating genes. These observations suggest the involvement of the GSL pathway in generating one or more negative effectors of growth that influence inflorescence architecture and fecundity by altering the balance of hormonal regulators.

Positive Impact of Multiple-Choice Question Authoring and Regular Quiz Participation on Student Learning.

We previously developed an online multiple-choice question authoring, learning, and self-assessment tool that we termed Quizzical. Here we report statistical analyses over two consecutive years of Quizzical use in a large sophomore-level introductory molecular biology course. Students were required to author two questions during the term and were also afforded opportunities to earn marks for quiz participation. We found that students whose final grade was "A," "B," or "C" exhibited similar patterns of Quizzical engagement. The degree to which students participated was positively associated with performance on formal exams, even if prior academic performance was considered as a covariable. During both terms investigated, students whose Quizzical engagement increased from one exam to the next earned statistically significant higher scores on the subsequent exam, and students who attempted Quizzical questions from earlier in the term scored higher, on average, on the cumulative portion of the final exam. We conclude that the structure and value of the assignment, and the utility of Quizzical as a discipline-independent active-learning and self-assessment tool, enabled students to better master course topics.

Digital Communication Technology: Does Offering a Choice of Modality Improve Medication Adherence and Outcomes in a Persistent Asthma Population?

BACKGROUND: Use of digital communication technology has shown potential to improve asthma adherence and outcomes. Few studies have looked at patient preference around mode of medication reminders used to improve and maintain asthma medication adherence. OBJECTIVE: To determine if, in a population already receiving automated medication reminders, offering a choice for preferred mode of reminder (text, email, phone) would improve their adherence and asthma outcomes over a 1-year period. METHODS: This was a pragmatic, randomized controlled trial conducted at Kaiser Permanente Colorado involving 7522 adult patients with persistent asthma. Study patients were randomized to receive usual care or their choice of medication reminder. Differences between the 2 groups in both medication adherence and asthma outcomes were then assessed over the following year. RESULTS: Only 30% of those offered a choice of medication reminder modality responded by making a choice, with 52% preferring text messaging. There was less of a decrease in adherence rate over the 1-year period in those who made a choice regarding the mode of medication refill reminder. There was no difference in asthma outcomes between those who did make a choice compared with those who did not make a choice regarding the mode of medication refill reminder. CONCLUSION: In a patient population already receiving medication reminders, offering a choice about what type of technology-enabled asthma medication reminder patients wanted did not improve outcomes but did enable a subgroup to better maintain their medication adherence.

Sensitivity of global soil carbon stocks to combined nutrient enrichment.

Soil stores approximately twice as much carbon as the atmosphere and fluctuations in the size of the soil carbon pool directly influence climate conditions. We used the Nutrient Network global change experiment to examine how anthropogenic nutrient enrichment might influence grassland soil carbon storage at a global scale. In isolation, enrichment of nitrogen and phosphorous had minimal impacts on soil carbon storage. However, when these nutrients were added in combination with potassium and micronutrients, soil carbon stocks changed considerably, with an average increase of 0.04 KgCm-2  year-1 (standard deviation 0.18 KgCm-2  year-1 ). These effects did not correlate with changes in primary productivity, suggesting that soil carbon decomposition may have been restricted. Although nutrient enrichment caused soil carbon gains most dry, sandy regions, considerable absolute losses of soil carbon may occur in high-latitude regions that store the majority of the world's soil carbon. These mechanistic insights into the sensitivity of grassland carbon stocks to nutrient enrichment can facilitate biochemical modelling efforts to project carbon cycling under future climate scenarios.

Noncatastrophic and catastrophic fractures in racing Thoroughbreds at the Hong Kong Jockey Club.

BACKGROUND: Reports of fractures in racehorses have predominantly focused on catastrophic injuries, and there are limited data identifying the location and incidence of fractures that did not result in a fatal outcome. OBJECTIVE: To describe the nature and the incidence of noncatastrophic and catastrophic fractures in Thoroughbreds racing at the Hong Kong Jockey Club (HKJC) over seven racing seasons. STUDY DESIGN: Retrospective cohort study. METHODS: Data of fractures sustained in horses while racing and of race characteristics were extracted from the HKJC Veterinary Management Information System (VMIS) and Racing Information System (RIS), respectively. The fracture event was determined from the first clinical entry for each specific injury. The incidence rates of noncatastrophic and catastrophic fractures were calculated per 1000 racing starts for racetrack, age, racing season, sex and trainer. RESULTS: One hundred and seventy-nine first fracture events occurred in 64,807 racing starts. The incidence rate of noncatastrophic fractures was 2.2 per 1000 racing starts and of catastrophic fractures was 0.6 per 1000 racing starts. Fractures of the proximal sesamoid bones represented 55% of all catastrophic fractures, while the most common noncatastrophic fractures involved the carpus and the first phalanx. Significant associations were detected between the incidence of noncatastrophic fractures and sex, trainer and racing season. MAIN LIMITATIONS: The first fracture event was used to calculate the incidence rate in this study and may have resulted in underestimation of the true incidence rate of fractures in this population. However, given the low number of recorded fracture events compared with the size of the study population, this underestimation is likely to be small. CONCLUSIONS: There were 3.6 times as many noncatastrophic fractures as catastrophic fractures in Thoroughbreds racing in Hong Kong between 2004 and 2011. Noncatastrophic fractures interfere with race training schedules and may predispose to catastrophic fracture. Future analytical studies on noncatastrophic racing fractures should be a priority for the racing industry.

Ultrasonographic-based predictive factors influencing successful return to racing after superficial digital flexor tendon injuries in flat racehorses: A retrospective cohort study in 469 Thoroughbred racehorses in Hong Kong.

BACKGROUND: Superficial digital flexor tendon (SDFT) injury is an important health and welfare concern in racehorses. It is generally diagnosed with ultrasonography, but predictive ultrasonographic features have not been reported. OBJECTIVES: To determine ultrasonographic features of forelimb SDFT injury at initial presentation in Thoroughbred racehorses that could predict a successful return to racing (completing ≥5 races). STUDY DESIGN: Retrospective cohort study. METHODS: Digitised ultrasonographic images of 469 horses with forelimb SDFT injuries from the Hong Kong Jockey Club (2003-2014) were evaluated, using a previously validated ultrasonographic scoring system. Six ultrasonographic parameters were evaluated (type and extent of the injury, location, echogenicity, cross-sectional area and longitudinal fibre pattern of the maximal injury zone [MIZ]), as well as horse signalment, retirement date and number of races before and after injury. Data were analysed by generalised linear regression with significance at P<0.05. RESULTS: Cases were divided into two groups: 1) For cases of SDFT tendonitis with core lesions, cross-sectional area at the MIZ was the most significant factor determining a successful return to racing (P = 0.03). If the lesion was <50% of the total cross-sectional area, horses had 29-35% probability of successfully racing again, but if it was ≥50% this decreased to 11-16%. 2) For cases of SDFT tendonitis without a core lesion, longitudinal fibre pattern at the MIZ best predicted a successful return to racing (P = 0.002); if the affected longitudinal fibre pattern was <75% of the total, horses had 49-99% probability of successfully return to racing, but if it was ≥75% this decreased to 14%. MAIN LIMITATIONS: Prognostic information may not be applicable to other breeds/disciplines. CONCLUSIONS: This is the first study to describe ultrasonographic features of forelimb SDFT injuries at initial presentation that were predictive of successful return to racing. The outcomes will assist with early, evidence-based decisions on prognosis in Thoroughbred racehorses.

Open standing castration in Thoroughbred racehorses in Hong Kong: Prevalence and severity of complications 30 days post-castration.

BACKGROUND: Complications following open standing castration (OSC) in Thoroughbred racehorses are well recognised but variation in their prevalence and severity between populations is not well documented. OBJECTIVES: To describe the prevalence and severity of complications in the 30 days following OSC. STUDY DESIGN: A retrospective cohort study of veterinary clinical records relating to horses that underwent OSC between July 2007 and July 2012. METHODS: Complications were graded on a severity score from N, no complications, to C3, severe complications. Additional data were accessed for each horse including age, import date, racing history, trainer and veterinarian performing the castration. Bacterial culture and antimicrobial sensitivities were performed on a limited number of castration wounds that became infected. RESULTS: In total, 250 horses were castrated in Hong Kong using the OSC technique over the period of the study. Sixty percent (150/250) of horses experienced some type of post-castration complication, with eight horses experiencing a severe (C3) complication requiring intensive veterinary treatment. Scrotal swelling, funiculitis and seroma formation were present in 70.0%, 36.7% and 24.7% of cases respectively. Most horses experiencing complications required wound reopening (87.3%; 131/150), and/or an extended course of first-line antimicrobials and/or nonsteroidal anti-inflammatory drugs (75/150; 50.0%). Eight horses had cultures submitted for bacterial sensitivity, with 17 bacterial isolates grown. In vitro, the bacteria cultured were sensitive to enrofloxacin (76%; 13/17) and ceftiofur (100%; 17/17). Resistance was detected to penicillin, gentamicin, oxytetracycline, metronidazole and trimethoprim-sulfadiazine. MAIN LIMITATIONS: Differences in post-castration management cannot be accounted for in this study. CONCLUSIONS: Complications following OSC in horses in Hong Kong were common. The majority were mild and were successfully treated using antimicrobials and simple wound management. Given the high rate of complications and antimicrobial usage identified in this study, a review of the technique is warranted.

Description of the incidence, clinical presentation and outcome of proximal limb and pelvic fractures in Hong Kong racehorses during 2003-2014.

BACKGROUND: Few studies have described incidences of proximal limb and pelvic fracture (PLPF) in Thoroughbred racehorses occurring on race day and during training. Information regarding clinical presentations and future racing careers in cases of PLPF is limited. OBJECTIVES: To describe the incidence, clinical presentation and outcome of PLPF sustained by horses in racing and training at the Hong Kong Jockey Club (HKJC) between 2003 and 2014. STUDY DESIGN: Retrospective cohort study. METHODS: Horses with PLPF confirmed by nuclear scintigraphy, ultrasonography, radiography or post-mortem examination were identified using veterinary clinical records. Training and racing data for case horses were described. Incidences of fractures were estimated per 1000 horses in training and per 1000 race starts for fractures sustained during racing. Descriptive statistics were used to describe the study population. RESULTS: A total of 129 instances of PLPF were sustained by 108 racehorses. The most commonly fractured bone was the humerus (49.6%), followed by the tibia (29.4%). Nine horses sustained fatal fractures, eight of which occurred during racing. The incidence of fracture during racing was 0.30 per 1000 starts. Two-thirds of fractures occurred during training. The majority of horses presented with grade 3 lameness (n = 42 of 119 injury events, 35.3%). All horses presenting with grade 5 lameness sustained fatal injuries. Following noncatastrophic injury, all horses underwent box rest and 81 horses subsequently resumed racing; 45 of these won a race. Horses were retired at a median of 25 months (interquartile range: 15-36 months) after injury. MAIN LIMITATIONS: Protocols for resting non-training racehorses at the HKJC and for recording rehabilitation regimens post-injury prevented the calculation of horse days at risk. CONCLUSIONS: The incidence of PLPF at the HKJC is low. Non-fatal PLPF is not necessarily a career-ending injury and many horses resume racing successfully following conservative treatment.

A novel Filamentous Flower mutant suppresses brevipedicellus developmental defects and modulates glucosinolate and auxin levels.

BREVIPEDICELLUS (BP) encodes a class-I KNOTTED1-like homeobox (KNOX) transcription factor that plays a critical role in conditioning a replication competent state in the apical meristem, and it also governs growth and cellular differentiation in internodes and pedicels. To search for factors that modify BP signaling, we conducted a suppressor screen on bp er (erecta) plants and identified a mutant that ameliorates many of the pleiotropic defects of the parent line. Map based cloning and complementation studies revealed that the defect lies in the FILAMENTOUS FLOWER (FIL) gene, a member of the YABBY family of transcriptional regulators that contribute to meristem organization and function, phyllotaxy, leaf and floral organ growth and polarity, and are also known to repress KNOX gene expression. Genetic and cytological analyses of the fil-10 suppressor line indicate that the role of FIL in promoting growth is independent of its previously characterized influences on meristem identity and lateral organ polarity, and likely occurs non-cell-autonomously from superior floral organs. Transcription profiling of inflorescences revealed that FIL downregulates numerous transcription factors which in turn may subordinately regulate inflorescence architecture. In addition, FIL, directly or indirectly, activates over a dozen genes involved in glucosinolate production in part by activating MYB28, a known activator of many aliphatic glucosinolate biosynthesis genes. In the bp er fil-10 suppressor mutant background, enhanced expression of CYP71A13, AMIDASE1 (AMI) and NITRILASE genes suggest that auxin levels can be modulated by shunting glucosinolate metabolites into the IAA biosynthetic pathway, and increased IAA levels in the bp er fil-10 suppressor accompany enhanced internode and pedicel elongation. We propose that FIL acts to oppose KNOX1 gene function through a complex regulatory network that involves changes in secondary metabolites and auxin.

Improved radiological diagnosis of palmar osteochondral disease in the Thoroughbred racehorse.

BACKGROUND: Palmar osteochondral disease (POD) is common in the Thoroughbred racehorse yet difficult to diagnose by radiography alone. OBJECTIVES: To improve the sensitivity and specificity for diagnosing POD from radiographs. STUDY DESIGN: Prospective, longitudinal study. METHODS: Radiographs in nine different projections were made of metacarpophalangeal joints (MCPJ) of 50 Thoroughbreds. Post-mortem, MCPJs were dissected and gross pathology was scored. Three experienced Thoroughbred clinicians read each radiograph and recorded their findings. Another clinician reviewed each radiograph alongside the related gross specimens in order to correlate radiological findings with joint pathology. This served as a 'gold standard'. The performance of each clinician at detecting POD was compared with the 'gold standard'. Radiological features associated with POD were identified and presented to the clinicians in a training manual, prior to them re-reading the radiographs. The ability of each clinician to diagnose POD was reassessed. RESULTS: Palmar osteochondral disease was a common finding (88/100 joints). All three clinicians demonstrated low sensitivity and low specificity at detecting POD (mean 0.37 and 0.75, respectively). Conversely, the sensitivity and specificity for POD in the 'gold standard' was high (0.95 and 1.0). POD was associated with primary radiological features (e.g. focal radiolucencies in the palmar condyles, disruption of the outline of subchondral bone and focal sclerosis of the palmar condyles) and secondary features (e.g. basilar and apical osteophytes on the proximal sesamoid bones, flattening of the palmar condyles and cavitation of the dorso-distal aspect of the third metacarpal bone). Secondary radiographic features were often easier to detect. Following training the performance of clinicians at identifying POD was significantly improved. MAIN LIMITATIONS: Only two clinicians completed the study; low proportion of POD negative cases in the study. CONCLUSIONS: Clinicians frequently overlook key radiological features related to POD. Alerting clinicians to relevant radiological features improved the sensitivity and specificity of diagnosis.

The incidence and risk factors for shipping fever in horses transported by air to Hong Kong: Results from a 2-year prospective study.

A 2 year prospective study was performed between February 2011 and January 2013 to determine the incidence and risk factors for shipping fever (SF) in horses transported by air to Hong Kong (HK). Using a questionnaire, data were collected from professional flying grooms regarding the journey to HK and horses in the shipment. Horses were monitored in quarantine for 2 weeks after arrival in HK, and clinical signs of SF recorded. Poisson and logistic regression models were used to identify risk factors for SF at the horse and shipment levels. The study analysed data from 869 horses on 81 flights arriving from Australia (n = 24), New Zealand (NZ; n = 18), the United Kingdom (UK; n = 33) and the United States of America (USA; n = 6). The incidence risk of SF was 10.8 per 100 horses and the proportion of shipments with at least one horse that developed SF was 49/81 (60%). The study identified that the rate per shipment of SF in shipments of horses originating from NZ, the USA and the UK was 2.40 (95% confidence interval [CI] 1.22-4.71), 2.43 (95% CI 0.66-8.89) and 3.08 (95% CI 1.60-5.93) times the rate of SF compared to Australia. Shipments arriving in HK during March and May were 5.61 (95% CI 1.55-20.31) and 4.51 (95% CI 1.43-14.26) times more likely to contain horses that developed SF compared to shipments arriving in January. The identification of these risk factors and the recognition of at-risk shipments will help focus attention on preventative strategies.

Impact of interactive voice response technology on primary adherence to bisphosphonate therapy: a randomized controlled trial.

UNLABELLED: Osteoporosis (weak bones) is a disorder that has high morbidity, mortality, and healthcare utilization. Effective treatment is available for this disorder, but many patients choose not to start therapy. This is the first study showing an intervention that increases the initiation rates to medications for osteoporosis. INTRODUCTION: One out of six patients prescribed an oral bisphosphonate does not initiate therapy, a phenomenon known as primary non-adherence. Reasons for bisphosphonate primary non-adherence have been identified, but not interventions that positively impact primary adherence rates. The purpose of this study is to determine the effectiveness of interactive voice response technology to improve oral bisphosphonate primary adherence. METHODS: This was a prospective, randomized controlled trial conducted in January-December 2014 at Kaiser Permanente Colorado, an integrated healthcare system. Adults with a new oral bisphosphonate prescription for osteoporosis or osteopenia which was not purchased within 14-20 days of being ordered were included. There were 127 and 118 patients in the intervention group and control groups, respectively. The intervention group received an interactive voice response phone call followed by a letter 1 week later if primary non-adherence continued, whereas the control group did not receive any outreach. The primary outcome was the proportion of patients who purchased their oral bisphosphonate within 25 days of randomization. RESULTS: There were 62/127 (48.8%) intervention patients and 36/118 (30.5%) control patients who purchased their bisphosphonate prescription within 25 days of randomization (OR = 2.17, 95% CI 1.29-3.67). When adjusted for age, sex, history of bone mineral density scan and fracture, the odds ratio for intervention versus control group was 2.3 (95% CI 1.34-3.94). CONCLUSION: An interactive voice response phone call and follow-up letter significantly improved primary adherence to oral bisphosphonate therapy. Such an intervention could be considered for improving primary adherence rates to other medication classes.

Comprehensive protein profiling of synovial fluid in osteoarthritis following protein equalization.

OBJECTIVE: The aim of the study was to characterise the protein complement of synovial fluid (SF) in health and osteoarthritis (OA) using liquid chromatography mass spectrometry (LC-MS/MS) following peptide-based depletion of high abundance proteins. DESIGN: SF was used from nine normal and nine OA Thoroughbred horses. Samples were analysed with LC-MS/MS using a NanoAcquity™ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMiner™. Progenesis-QI™ LC-MS software was used for label-free quantification. In addition immunohistochemistry, western blotting and mRNA expression analysis was undertaken on selected joint tissues. RESULTS: The number of protein identifications was increased by 33% in the ProteoMiner™ treated SF compared to undepleted SF. A total of 764 proteins (462 with≥2 significant peptides) were identified in SF. A subset of 10 proteins were identified which were differentially expressed in OA SF. S100-A10, a calcium binding protein was upregulated in OA and validated with western blotting and immunohistochemistry. Several new OA specific peptide fragments (neopeptides) were identified. CONCLUSION: The protein equalisation method compressed the dynamic range of the synovial proteins identifying the most comprehensive SF proteome to date. A number of proteins were identified for the first time in SF which may be involved in the pathogenesis of OA. We identified a distinct set of proteins and neopeptides that may act as potential biomarkers to distinguish between normal and OA joints.

Descriptive analysis of longitudinal endoscopy for exercise-induced pulmonary haemorrhage in Thoroughbred racehorses training and racing at the Hong Kong Jockey Club.

REASONS FOR PERFORMING STUDY: Exercise-induced pulmonary haemorrhage (EIPH) is reported as a performance limiting condition in racehorses, yet few longitudinal studies characterising EIPH have been reported. OBJECTIVES: To characterise EIPH during training and racing in the absence of prophylactic medication with furosemide among horses imported to Hong Kong during 2007-2012. STUDY DESIGN: Retrospective descriptive study of clinical endoscopy, EIPH status, and racing records. METHODS: Thoroughbred geldings (n = 822) imported from New Zealand between 2007 and 2012 were retrospectively assigned to 4 groups: diagnosed with EIPH via endoscopy (EIPH+), graded using recognised criteria; observed with epistaxis (Epistaxis); free of EIPH on endoscopy (EIPH-); and those in which no endoscopy was performed. RESULTS: The majority of horses (89%) were subjected to endoscopy once or more (median 15, interquartile range 3-18). Of those undergoing endoscopy, 55% of horses were diagnosed EIPH+ which varied in severity. Few (4%) experienced epistaxis. EIPH+ was diagnosed most frequently (63%) after racing. There was no significant difference in the proportion of EIPH+ and EIPH- horses that raced. Racing career longevity was longest for EIPH+ horses. The number of starts in Hong Kong for EIPH+ horses was not significantly different to EIPH- horses. [Correction added on 9 January 2015, after first online publication: The term 'lifetime starts' has been changed to 'starts in Hong Kong' in the preceding sentence.] Days to retirement were longer for EIPH+ horses. Horses with mild EIPH+ (grade <3) were more likely to be retired for other causes whereas severe grades (≥3) were more likely to be retired for EIPH. CONCLUSIONS: Exercise-induced pulmonary haemorrhage is common and varies in severity between individuals and between episodes in the same individual. There is no difference in racing career longevity between EIPH+ and EIPH- horses trained and raced without furosemide. See also correspondences by PS Morley and KW Hinchcliff; AD Richards; and S. Preston and C. M. Riggs.

Pharmacokinetic evaluation of oral dantrolene in the dog.

The pharmacokinetics of dantrolene and its active metabolite, 5-hydroxydantrolene, after a single oral dose of either 5 or 10 mg/kg of dantrolene was determined. The effects of exposure to dantrolene and 5-hydroxydantrolene on activated whole-blood gene expression of the cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) were also investigated. When dantrolene was administered at a 5 mg/kg dose, peak plasma concentration (Cmax ) was 0.43 µg/mL, terminal half-life (t1/2 ) was 1.26 h, and area under the time-concentration curve (AUC) was 3.87 µg·h/mL. For the 10 mg/kg dose, Cmax was 0.65 µg/mL, t1/2 was 1.21 h, and AUC was 5.94 µg·h/mL. For all calculated parameters, however, there were large standard deviations and wide ranges noted between and within individual dogs: t1/2 , for example, ranged from 0.43 to 6.93 h, Cmax ratios ranged from 1.05 to 3.39, and relative bioavailability (rF) values ranged from 0.02 to 1.56. While activated whole-blood expression of IL-2 and IFN-γ as measured by qRT-PCR was markedly suppressed following exposure to very high concentrations (30 and 50 µg/mL, respectively) of both dantrolene and 5-hydroxydantrolene, biologically and therapeutically relevant suppression of cytokine expression did not occur at the much lower drug concentrations achieved with oral dantrolene dosing.

Characterization of a cruciferin deficient mutant of Arabidopsis and its utility for overexpression of foreign proteins in plants.

Plant seeds naturally accumulate storage reserves (proteins, carbohydrates, lipids) that are mobilized during germination to provide energy and raw materials to support early seedling growth. Seeds have been exploited as bioreactors for the production to foreign materials, but stable, high level expression has been elusive, in part due to the intrinsic bias for producing the natural reserves in their typical proportions. To identify mutants governing seed filling, we screened a population of mutagenized Arabidopsis plants for a mutant that failed to fill its seeds. Here we report the identification of ssp1, a recessive, viable mutant that accumulates approximately 15% less protein than wildtype seeds. Molecular analyses revealed that ssp1 is due to the introduction of a premature stop codon in CRU3, one of the major cruciferin genes. Unlike many other reserve mutants or transgenic lines in which seed storage protein levels are reduced by antisense/RNAi technologies, ssp1 exhibits low level compensation by other reserves, and represents a mutant background that might prove useful for high level expression of foreign proteins. To test this hypothesis, we used a bean phytohemagglutinin (PHA) gene as a reporter and compared PHA expression levels in single copy insertion lines in ssp1 vs. wildtype. These near isogenic lines allow reporter protein levels to be compared without the confounding and sometimes unknown influences of transgene copy number and position effects on gene expression. The ssp1 lines consistently accumulated more PHA than the backcrossed counterparts, with increases ranging from 12% to 126%. This proof of principle study suggests that similar strategies in crop plants may improve the yield of foreign proteins of agronomic and economic interest.